Diagnosis of candidiasis and candidemia or invasive candida infection

ABSTRACT

The use of an antibody to a  C albicans  cell wall antigen or to a solubilized phosphopeptidomannan (PPM) fraction of the cell wall of  C albicans , or preferably a combination of an IgG2 antibody to a phosphopeptidomannan (PPM) fraction of the cell wall of  C albicans , and an IgG1 or IgG3 antibody to a  C albicans  cell wall antigen in the diagnosis of candidiasis or invasive candidiasis is disclosed. Also diagnostic tests are disclosed.

FIELD OF THE INVENTION

The present invention relates to the use of Candida albicans cell wallantigens for serological diagnosis of systemic candidiasis, includingIgG sub-class antibodies to C albicans cell wall antigens.

BACKGROUND OF THE INVENTION

Invasive candidosis is difficult to diagnose and is the cause ofsubstantial morbidity and mortality in immunosuppressed patients. Theclinical symptoms are often vague and conventional blood culturing isoften negative [de Repentigny L, Kuykendall R. J, Chandler F. W,Broderson J. R, Reiss E. Comparison of serum mannan, arabinitol, andmannose in experimental disseminated candidiasis. J Clin Microbiol,1984. 19(6): p. 804-12; Gutierrez J, Maroto C, Piedrola G, Martin E,Perez J. A. Circulating Candida antigens and antibodies: useful markersof candidemia. J Clin Microbiol, 1993. 31(9): p. 2550-2; Jones, J. M.,Laboratory diagnosis of invasive candidiasis. Clin Microbiol Rev, 1990.3(1): p. 32-45]. Laboratory diagnosis of deep Candida infection is basedon blood culturing, direct microscopy, and determination of Candidaantigens. Anti-Candida antibodies and detection of D-arabinitol in serumor urine by gas liquid chromatography are complementary analyses [deRepentigny L, Kuykendall R. J, Chandler F. W, Broderson J. R, Reiss E.Comparison of serum mannan, arabinitol, and mannose in experimentaldisseminated candidiasis. J Clin Microbiol, 1984. 19(6): p. 804-12;Gutierrez J, Maroto C, Piedrola G, Martin E, Perez J. A. CirculatingCandida antigens and antibodies: useful markers of candidemia. J ClinMicrobiol, 1993. 31(9): p. 2550-2; Fujita, S, Hashimoto T, Detection ofserum Candida antigens by enzyme-linked immunosorbent assay and a latexagglutination test with anti-Candida albicans and anti-Candida kruseiantibodies. J Clin Microbiol, 1992. 30(12): p. 3132-7; Nakamura A,Ishikawa N, Suzuki H. Diagnosis of invasive candidiasis by detection ofmannan antigen by using the avidin-biotin enzyme immunoassay. J ClinMicrobiol, 1991. 29(11): p. 2363-7].

Antibody determination for immunodiagnosis of systemic candidosisincludes latex agglutination [Dee T. H., Johnson G. M, Berger C. S.Sensitivity, specificity, and predictive value of anti-candida serumprecipitin and agglutinin quantification: comparison ofcounterimmunoelectrophoresis and latex agglutination. J Clin Microbiol,1981. 13(4): p. 750-3], counterimmunoelectrophoresis [Bisbe J, Miro J.M, Torres J. M, Latorre X, Alia C, Amaral M, Estivill D, Mallolas J,Trilla A, Soriano E. Diagnostic value of serum antibody and antigendetection in heroin addicts with systemic candidiasis. Rev Infect Dis,1989. 11(2): p. 310-5; Kostiala, A. A. and I. Kostiala. Enzyme-linkedimmunosorbent assay (ELISA) for IgM, IgG and IgA class antibodiesagainst Candida albicans antigens: development and comparison with othermethods. Sabouraudia, 1981. 19(2): p. 123-34], indirectimmunofluorescence [Quindos G, Ponton J, Cisterna R. Detection ofantibodies to Candida albicans germ tube in the diagnosis of systemiccandidiasis. Eur J Clin Microbiol, 1987. 6(2): p. 142-6] andenzyme-linked immunoassay [Kostiala, A. A. and I. Kostiala.Enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA classantibodies against Candida albicans antigens: development and comparisonwith other methods. Sabouraudia, 1981. 19(2): p. 123-34; Greenfield R.A., Bussey, M. J, Stephens J. L, Jones J. M. Serial enzyme-linkedimmunosorbent assays for antibody to Candida antigens during inductionchemotherapy for acute leukemia. J Infect Dis, 1983. 148(2): p. 275-83;Navarro D, Monzonis E., Lopez-Ribot J. L, Sepulveda P, Casanova M,Nogueira J. M, Martinez J. P. Diagnosis of systemic candidiasis byenzyme immunoassay detection of specific antibodies to mycelial phasecell wall and cytoplasmic candidal antigens. Eur J Clin Microbiol InfectDis, 1993. 12(11): p. 839-46]. The antibody tests often express a lowsensitivity, i.e. in most cases they fail to discriminate betweendisseminated and superficial candidosis [Martinez J. P, Gil M. L,Lopez-Ribot J. L., Chaffin W. L. Serologic response to cell wallmannoproteins and proteins of Candida albicans. Clin Microbiol Rev,1998. 11(1): p. 121-41]. In order to increase the specificity of theimmundiagnostic tests, various forms of antigens from C. albicansincluding cytoplasmic extract and cell wall antigens [Sendid, B,Tabouret M., Poirot J. L, Mathieu D, Fruit J, Poulain D. New enzymeimmunoassays for sensitive detection of circulating Candida albicansmannan and antimannan antibodies: useful combined test for diagnosis ofsystemic candidiasis. J Clin Microbiol, 1999. 37(5): p. 1510-7; ZollerL, Kramer I, Kappe R, Sonntag H. G. Enzyme immunoassays for invasiveCandida infections: reactivity of somatic antigens of Candida albicans.J Clin Microbiol, 1991. 29(9): p. 1860-7] have been used.

The cell wall of C albicans makes up approximately 30% of the totalweight of the cell and is composed of carbohydrate (glucan, mannan, andchitin), small amounts of protein, and lipids. The outermost layer ofthe cell wall is composed of mannoprotein while the deeper layer is madeup of β-glucan and chitin microfibrils [Reiss E., Hearn V. M, Poulain D,Shepherd M. G. Structure and function of the fungal cell wall. J Med VetMycol, 1992. 30(Suppl 1): p. 143-56]. Mannoprotein is a complexglycoprotein composed of mannose polymers and oligomers attached invarious ways to a peptide. Mannose or unbranched mannose oligomers arealso attached directly to a peptide via the hydroxyl groups of serine orthreonine residues [Nelson R. D, Shibata N, Podzorski R. P, Herron M. J.Candida mannan: chemistry, suppression of cell-mediated immunity, andpossible mechanisms of action. Clin Microbiol Rev, 1991. 4(1): p. 1-19].

Anti-mannan antibodies appear to be one of the major anti-Candidaantibodies in human sera [Jones J. M. Quantitation of antibody againstcell wall mannan and a major cytoplasmic antigen of Candida in rabbits,mice, and humans. Infect Immun, 1980. 30(1): p. 78-89]. The glucanpolymers which are in greater abundance than mannan in the C. albicanscell wall are immunologically less active [Nelson R. D, Shibata N,Podzorski R. P, Herron M. J. Candida mannan: chemistry, suppression ofcell-mediated immunity, and possible mechanisms of action. ClinMicrobiol Rev, 1991. 4(1): p. 1-19]. Since mannan is a major antigeniccomponent of the cell wall, different chemical and enzymatic methodshave been used to extract mannan from C. albicans, but these methodshave limitations. When mannan is extracted by hot alkali treatment,mannose-serine, and mannose-threonine linkages, phosphodiester linkagesand some peptide bonds are cleaved at basic pH. The loss of the O-linkedoligosaccharides attached via phosphate groups leaves a modified mannanproduct that is greatly altered in its antigenicity and biologicaleffects [Nelson R. D, Shibata N, Podzorski R. P, Herron M. J. Candidamannan: chemistry, suppression of cell-mediated immunity, and possiblemechanisms of action. Clin Microbiol Rev, 1991. 4(1): p. 1-19]. Hotwater extraction may denature the protein structure in mannoproteins.Thus the extraction procedures may modify the mannan product and alterthe antigenicity.

Antibody tests have been less useful mainly because anti-Candidaantibodies are often present in healthy individuals which is consideredto be a consequence of immunization from Candida in the commensal flora(Odds, F. C., and E. G. Evans. 1980. Distribution of pathogenic yeastsand humoral antibodies to candida among hospital inpatients. J ClinPathol 33:750-6). Furthermore, the antibody response of Candida-infectedpatients is often impaired by the underlying condition, particularly innon-surgical patients.

The human IgG subclasses differ with respect to physical, chemical, andbiological properties. IgG1, IgG2, and IgG3 activate complement,although IgG2 less efficiently. In contrast to IgG1, IgG3, and IgG4,IgG2 demonstrates no or low binding capacity to human mononuclear cellsand neutrophils. The subclass distribution of the antibody response isinfluenced by the nature of the immunogen, the localization of the entryinto the body, and age of the host. IgG1 and IgG3 antibodies are mainlyinduced by protein antigens whereas IgG2 is predominating againstpolysaccharides (Hammarstrom, L., M. Granstrom, V. Oxelius, M. A.Persson, and C. I. Smith. 1984. IgG subclass distribution of antibodiesagainst S. aureus teichoic acid and alpha-toxin in normal andimmunodeficient donors. Clin Exp Immunol 55:593-601; Mattsby-Baltzer,I., L. Edebo, B. Jarvholm, B. Lavenius, and T. Soderstrom. 1990.Subclass distribution of IgG and IgA antibody response to Pseudomonaspseudoalcaligenes in humans exposed to infected metal-working fluid. JAllergy Clin Immunol 86:231-8; Shakib, F., and D. R. Stanworth. 1980.Human IgG subclasses in health and disease. (A review). Part I. Ric ClinLab 10:463-79).

DESCRIPTION OF THE INVENTION

One object of the present invention is to provide more native forms ofC. albicans cell wall antigens which may improve the serological testfor identifying patients with candidosis.

The present invention relates to the use of an antibody to a C albicanscell wall antigen, or to a solubilized phosphopeptidomannan (PPM)fraction of the cell wall of C albicans, or preferably a combination ofan IgG2 antibody to a phosphopeptidomannan (PPM) fraction of the cellwall of C albicans, and an IgG1 or IgG3 antibody to a C albicans cellwall antigen in the diagnosis of candidiasis or invasive candidiasis.

In view of the above mentioned problems, an attempt was made to preparenative cell wall fragments and to release the phosphopeptidomannan fromC. albicans cell wall by a gentle method to keep the immunogenic regionsmore intact. The antibody activity against various C albicans cell wallpreparations (cell wall fragments, modified cell wall fragments,phosphopeptidomannan, and β(1-3)(1-6) glucan) was investigated in orderto find antigens with high discriminatory power for diagnosis of acutedeep Candida infection. Rabbit immune sera and sera from patients withcandidemia were used for this purpose. The antibody levels weredetermined by the enzyme linked immunosorbent assay (ELISA).

Serological tests for diagnosis of disseminated fungal infections in theimmunocompromised host are used with varying results. In the workleading to the present invention, the discriminatory power of antibodiesto C albicans cell wall components was evaluated in order to findantigenic markers for serological diagnosis of candidemia.

Native C albicans cell wall fragments (CW), periodate (CW_(IO4)) andproteinase-K (CW_(P)) treated CW, a mildly extractedphosphopeptidomannan (PPM), and β(1-3)(1-6)-glucan were used as antigensin ELISA with sera from rabbits immunized with C albicans (n=10),patients with culture proven candidemia (n=8), and healthy individuals(n=8).

The antibody response in rabbits was predominated by anti-PPM antibodiesa finding which was confirmed by inhibition-ELISA. Accordingly,periodate treatment (CW_(IO4)) destroyed a major part of the antigenicepitopes. Low rabbit antibody levels were found against glucan, themajor Candida cell wall component supporting its localization mainly inthe inner part of the C albicans cell wall. Somewhat different from therabbit serum IgG antibody response against PPM, which was at leasttenfold higher than against CW, the human IgG antibody levels inpatients with candidemia were similar for both antigens, the levelsbeing significantly higher than in the healthy controls (CW, P=0.0005and PPM, P<0.0001). Although the human anti-glucan and anti-CW_(IO4) IgGantibody levels were overall low, they were still significantlyincreased in the patient group (P=0.0159 and P=0.0491, respectively). Inaddition a correlation was noticed between these antibodies. Nosignificant differences were found for IgM antibodies between patientsand controls using CW, CW_(IO4), PPM and Glu as antigens. In conclusion,IgG antibodies to PPM and native cell wall fragments (CW) were highlydiscriminatory for candidemia and thus promising antigen candidates forserodiagnosis.

Sera from a total of 15 patients collected on two to three consecutiveoccasions starting at the day of culture-proven candidiasis wereanalyzed by enzyme linked immunosorbent assay (ELISA). Control groupsconsisted of lactating mothers (n=9, group I) with breast milk positivefor C. albicans and acute inflammation of the nipples and age-matchedblood donors (n=10, group II).

It was shown that within the first three weeks all patients werepositive for β(1-3)-glucan (Gluspecy), but none for mannan (PastorexCandida). The controls were neither positive for β(1-3)-glucan (<20βg/ml) nor mannan (<2.5 ng/ml). IgG1 anti-CW and IgG2 anti-PPMantibodies were the most discriminatory antibodies. The IgG1 anti-CWantibodies were partly less sensitive to non-C. albicans candidiasis.The ratios of IgG1 anti-CW/IgG2 anti-PPM were significantly lower innon-surviving patients than in the other patients within the first 6days of candidiasis (P=0.019). IgG2 anti-CW_(IO4) and anti-glucanantibodies correlated strongly (p<0.0001) and a lack of these antibodieswere associated with increasing β(1-3)-glucan levels. A high antibodylevel for IgG1 anti-CW or IgG2 anti-PPM antibodies (≧log 3), or the sumof them (≧log 5) showed a 92% sensitivity and 100% specificity andpositive predictive value. In conclusion, β(1-3)-glucan and the twosubclass antibodies seem to be early specific markers in the laboratorydiagnosis of candidiasis. The kinetics of the β(1-3)-glucan level mayalso help in evaluating the therapeutic efficacy of antimycotictreatment.

Thus, the present invention relates to the following:

The use of a combination of an IgG2 antibody to a phosphopeptidomannan(PPM) fraction of the cell wall of C albicans, and an IgG1 antibody to aC albicans cell wall antigen, and glucan for the diagnosis ofcandidiasis or invasive candidiasis.

The use of an antibody, such as an IgG2 antibody, an IgG1 antibodyand/or an IgG3 antibody, to a C albicans cell wall antigen or to asolubilized phosphopeptidomannan (PPM) fraction of the cell wall of Calbicans for the diagnosis of candidiasis or invasive candidiasis.

A diagnostic kit for the diagnosis of candidiasis or invasivecandidiasis comprising

means for drawing a sample from a patient;

means for an assay, such as a a sandwich ELISA assay, for the detectionof a combination of an IgG2 antibody to a phosphopeptidomannan (PPM)fraction of the cell wall of C albicans, and an IgG1 antibody to a Calbicans cell wall antigen, and glucan, wherein said sample is analyzedfor the presence of the simultaneous presence of an IgG2 antibody to aphosphopeptidomannan (PPM) fraction of the cell wall of C albicans, andan IgG1 antibody to a C albicans cell wall antigen, and glucan.

Diagnostic kit for the diagnosis of candidiasis or invasive candidiasiscomprising

means for drawing a sample from a patient;

means for an assay, such as a sandwich ELISA assay, for the detection ofan antibody, such as an IgG2 antibody, an IgG1 antibody and/or an IgG3antibody, to a C albicans cell wall antigen or to a solubilizedphosphopeptidomannan (PPM) fraction of the cell wall of C albicans,wherein said sample is analyzed for the presence of an antibody to a Calbicans cell wall antigen or to a solubilized phosphopeptidomannan(PPM) fraction of the cell wall of C albicans.

A method for diagnosing candidiasis or invasive candidiasis a patientcomprising

drawing a sample from the patient, and

performing an assay for the detection of an IgG2 antibody to aphosphopeptidomannan (PPM) fraction of the cell wall of C albicans, andan IgG1 antibody to a C albicans cell wall antigen, and glucan, whereinthe simultaneous presence of an IgG2 antibody to a phosphopeptidomannan(PPM) fraction of the cell wall of C albicans, and an IgG1 antibody to aC albicans cell wall antigen, and glucan indicates candidiasis orinvasive candidiasis in the patient

A method for diagnosing candidiasis or invasive candidiasis a patientcomprising

drawing a sample from the patient, and

performing an assay for the detection of an antibody to a C albicanscell wall antigen or to a solubilized phosphopeptidomannan (PPM)fraction of the cell wall of C albicans, wherein the presence of anantibody to a C albicans cell wall antigen or to a solubilizedphosphopeptidomannan (PPM) fraction of the cell wall of C albicansindicates candidiasis or invasive candidiasis in the patient.

The use of an antibody, such as an IgG antibody to a native cell wallfragment of C albicans and/or an IgG antibody to a solubilizedphosphopeptidomannan (PPM) fraction of the cell wall of C albicans,preferably a human serum IgG antibody, for the diagnosis of candidemiaor invasive Candida infection.

Diagnostic kit for the diagnosis of candidemia or invasive Candidainfection comprising

means for drawing a sample from a patient;

means for an assay, such a sandwich ELISA assay, for the detection of anIgG antibody, such as a human serum IgG antibody, to a native cell wallfragment of C albicans or an IgG antibody to a solubilizedphosphopeptidomannan (PPM) fraction of the cell wall of C albicans,wherein said sample is analyzed for the presence of an IgG antibody to anative cell wall fragment of C albicans or an IgG antibody to asolubilized phosphopeptidomannan (PPM) fraction of the cell wall of Calbicans.

A method for diagnosing candidemia or invasive Candida infection in apatient comprising

drawing a sample from the patient, and

performing an assay for the detection of an IgG antibody, such as ahuman serum IgG antibody, to a native cell wall fragment of C albicansor an IgG antibody to a solubilized phosphopeptidomannan (PPM) fractionof the cell wall of C albicans, wherein the presence of an IgG antibodyto a native cell wall fragment of C albicans or an IgG antibody to asolubilized phosphopeptidomannan (PPM) fraction of the cell wall of Calbicans indicates candidemia or invasive Candida infection in thepatient.

The invention will now be further explained in the following examples.These examples are only intended to illustrate the invention and shouldin no way be considered to limit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the examples, reference is made to the accompanying drawings onwhich:

FIG. 1 is a microphotograph of C albicans yeast cells treated with glassbeads (phase contrast microscopy, magnification 500). Dead cells arepresented by dark empty sacs, while whole yeast cells appear with brightcenters.

FIG. 2 illustrate. IgG and IgM antibodies to CW in five rabbitsimmunized with formalin killed yeast cells (σ, λ) and CW (ν, υ, τ) asanalyzed by ELISA. Serum samples were also collected before the start ofimmunization (day 0). The antibody concentrations are expressed as theabsorbance value at a serum dilution of 1/1000.

FIG. 3 illustrates rabbit IgG anti-C albicans antibodies analyzed byELISA using CW, CW_(P), CW_(IO4), PPM, Glu and Man as antigens. The serawere analyzed at a dilution of 1/100.

FIG. 4 illustrates how the rabbit serum pool (dilution 1/2000) wasabsorbed with varying concentrations of antigens and analyzed forremaining IgG and IgM antibody activities against CW. Absorption wasperformed with CW (λ), CW_(IO4) (ν) and CW_(P) (σ). The antibodyactivity of the unabsorbed serum pool is indicated.

FIG. 5 illustrates serum IgG and IgM antibodies to CW, CW_(IO4), PPM,and Glu in patients with culture-proven candidiasis (PS) and healthyblood donors (C) as analyzed by ELISA. Median values for each group arepresented. Statistical comparison was performed with Welch t test.

FIG. 6 illustrates glucan concentration in serum from patients withsystemic candidiasis, superficial Candida infection (control group I),and healthy blood donors (group II) (a). Kinetics of the glucanconcentration for each patient with respect to the time at which Candidawas isolated (day 0) (b). The detection limit was 10 pg/ml as determinedby Gluspecy.

FIG. 7 illustrates serum IgG1, IgG2, and IgG3 antibodies to C albicanscell wall antigens in patients with systemic candidiasis and controls(group I and II). The antibody activity was analysed by ELISA (<2 log,no antibody activity in serum diluted 1:100).

FIG. 8 illustrates IgG1, IgG2, and IgG3 antibodies to C albicans cellwall antigens in patients with systemic candidiasis at different timepoints after isolation of Candida from the patient. The antibodyactivity was analyzed by ELISA. Dashed lines indicate patients withnon-albicans infections, open symbols indicate patients who did notsurvive the infection.

EXPERIMENTAL—PART I Materials and Methods

Antigens

C. albicans (strain ATCC 64549) serotype A was grown in Sabouraddextrose broth on a shaker at 37° C., for 24 h. Yeast cells wereobtained from the culture medium by centrifugation. The antigenicfactors 4, 5 and 6 were expressed by the yeast cells as determined byCandida Check (Iatron laboratories, Japan), an agglutination test usingrabbit polyclonal antibodies directed to factor 4, 5 and 6.

Formalinized yeast cells were used for immunization of rabbits. Thus,cells were suspended in formaldehyde (5%) and kept at r.t. overnight.The cells were washed two times with distilled water and once with PBS.Before the cells were lyophilized the killing efficiency was checked byculturing.

Cell wall fragments of C albicans (CW). After repeated washings of thecultivated cells, the cell pellet was suspended in an equal volume ofdistilled water. Glass beads (0.5 mm in diameter) were added to the cellsuspension in a volume ratio of 1:3. The mixture was shaken repeatedly(20 cycles) in a vortex mixer for one minute with intervals on ice. Thesupernatant was collected and the glass beads were washed several timeswith distilled water, till the washing solution became clear. Thesupernatant and washing solutions were pooled. The cell wall fractionwas sedimented at 1200×g for 10 min and washed 10 times with ice-colddistilled water. Almost complete cell breakage (90-95%) was obtained asassessed by phase-contrast microscopy (FIG. 1). The CW suspension waswashed three times with distilled water and freeze-dried.

Modified CW. CW was treated with sodium periodate in order to destroythe carbohydrate structures by oxidation. To 16 mg of lyophilized CWsuspended in 5 ml of 0.1M sodium phosphate buffer (pH 6) was added 0.5ml of 0.15 M NaIO₄ [Quash G, Roch A. M, Niveleau A, Grange J,Keolouangkhot T, Huppert J. The preparation of latex particles withcovalently bound polyamines, IgG and measles agglutinins and their usein visual agglutination tests. J Immunol Methods, 1978. 22(1-2): p.165-74]. The mixture was left at 4° C. in the dark for 3 h. The reactionwas stopped by adding 0.6 ml of 0.15 M Na₂SO₃. After 30 min, the mixturewas dialyzed against 0.1 M phosphate buffer pH 6, at 4° C. overnight.The buffer was changed several times to remove low molecular weightoxidation products. Finally the CW was dialyzed against distilled water.The periodate treated CW suspension (CW_(IO4)) was lyophilized.

CW was also treated with proteinase K, in order to reduce the proteincontent. Lyophilized CW (20 mg/ml) was incubated with proteinase K(final concentration 2 mg/ml) (Sigma, USA), at 37° C. for 3 h [HitchcockP. J, Brown T. M. Morphological heterogeneity among Salmonellalipopolysaccharide chemotypes in silver-stained polyacrylamide gels. JBacteriol, 1983. 154(1): p. 269-77]. Thereafter the enzyme washeat-inactivated by incubation at 70° C. for 30 min. The suspension waswashed three times with distilled water and finally dialyzed at 4° C.overnight. The dialysate (CW_(P)) was lyophilized.

Phosphopeptidomannan (PPM). PPM was extracted by a method used by Lloydwith Cladosporium werneckii [Lloyd K. O. Isolation, characterization,and partial structure of peptido galactomannans from the yeast form ofCladosporium werneckii. Biochemistry, 1970. 9(17): p. 3446-53].Freeze-dried C albicans yeast cells (11 g) were suspended in 110 ml of0.05 M potassium phosphate buffer (pH 7.0) and heat-treated at 100° C.for 2 hr. After cooling the extract was fractionated with 0.22 g ofhexadecyltrimethylammonium bromide (Cetavlon) at pH 8.8, in the presenceof 6.1 ml of boric acid (1%). The precipitate was centrifuged and washedwith 0.5% sodium borate (pH 8.8). The residue was dissolved in 2% aceticacid (3.1 ml) and sodium acetate (0.06 g). Ethanol (3 volumes) was addedto the solution. The resulting precipitate was removed by centrifugationand washed with 2% acetic acid in ethanol followed only by ethanol. Theproduct was lyophilized and yielded 0.108 g (1%) of material. The PPMwas highly water soluble.

PPM was further characterized by SDS polyacrylamide gel electrophoresis(3-8% gradient gel, Invitrogen life technologies, USA). PPM was mixed insample buffer (NuPage sample buffer, Invitrogen,) and heat treated at70° C. for 10 min prior to application onto the gel. The gels werestained for protein by Silver Stain Plus (BIORAD, USA) and forcarbohydrate by the periodic acid-Schiff(PAS) method [Kapitany R. A,.Zebrowski E. J. A high resolution PAS stain for polyacrylamide gelelectrophoresis. Anal Biochem, 1973. 56(2): p. 361-9].

Degradation of PPM with acid and alkali. In order to characterize acidand/or alkali-labile epitopes, PPM was treated with HCl and NaOH asdescribed by Kobayashi et. al [Kobayashi H, Matsuda K, Ikeda T, SuzukiM, Takahashi S, Suzuki A, Shibata N, Suzuki S. Structures of cell wallmannans of pathogenic Candida tropicalis IFO 0199 and IFO 1647 yeaststrains. Infect Immun, 1994. 62(2): p. 615-22]. PPM was dissolved in 10mM HCl in a final concentration of 9 mg/ml and incubated at 100° C. for1 h as described by Kobayashi [Kobayashi H, Matsuda K, Ikeda T, SuzukiM, Takahashi S, Suzuki A, Shibata N, Suzuki S. Structures of cell wallmannans of pathogenic Candida tropicalis IFO 0199 and IFO 1647 yeaststrains. Infect Immun, 1994. 62(2): p. 615-22]. The solution wasneutralized with 100 mM NaOH. The acid treated PPM was designatedPPM_(HCl). For alkali treatment, the PPM was dissolved in 100 mM NaOH ata final concentration of 6.7 mg/ml. The resultant solution was kept at25° C. for 18 h and thereafter neutralized with 1 M HCl [Kobayashi H,Matsuda K, Ikeda T, Suzuki M, Takahashi S, Suzuki A, Shibata N, SuzukiS. Structures of cell wall mannans of pathogenic Candida tropicalis IFO0199 and IFO 1647 yeast strains. Infect Immun, 1994. 62(2): p. 615-22].The alkali treated PPM was designated PPM_(NaOH).

Glucan (Glu). Glu prepared from Saccharomyces cerevisiae (Sigma, StLouis), barley (Glu-B), and Alcaligenes faecalis (Glu-C) was used. Gluconsists of β(1-3)(1-6)-D-linked glucose residues, which is also acomponent of C. albicans glucan [Kapteyn J. C, Montijn R. C, DijkgraafG. J, Van den Ende H, Klis F. M. Covalent association of beta-1,3-glucanwith beta-1,6-glucosylated mannoproteins in cell walls of Candidaalbicans. J Bacteriol, 1995. 177(13): p. 3788-92; Sanjuan R., Zueco J,Stock R, Font de Mora J, Sentandreu R. Identification ofglucan-mannoprotein complexes in the cell wall of Candida albicans usinga monoclonal antibody that reacts with a (1,6)-beta-glucan epitope.Microbiology, 1995. 141(Pt 7): p. 1545-51; Smits G. J, Kapteyn J. C, vanden Ende H, Klis F. M. Cell wall dynamics in yeast Curr Opin Microbiol,1999. 2(4): p. 348-52]. Glu-B consists of β(1-4)(1-6)-D-linked glucoseresidues. Glu-C consists entirely of β(1-3)-D-linked glucose residues.Glu and Glu-C, which are insoluble in water, were solubilized in 0.1 MNaOH at a concentration of 10 mg/ml. These solutions were then used forchemical analyses.

Mannan (Man). Mannan of C albicans ATCC 64549 was prepared in accordancewith the description of Koucorek and Bellou [Kocourek J, Ballou C. E.Method for fingerprinting yeast cell wall mannans. J Bacteriol, 1969.100(3): p. 1175-81]. Briefly, mannan was extracted in 0.02 M citratebuffer (pH 7.0) at 125° for 90 min, followed by precipitation withFehling solution. The copper complex was dissolved in 3 M HCl, andprecipitated with methanol-acetic acid, then washed with ethanol andethyl-ether. The product was air dried.

This antigen was prepared in order to compare the commonly used mannanextraction procedure with that of PPM.

Determination of Protein, Hexose and Phosphate

The protein content of the various antigens was determined by acolorimetric method. A set of standard protein concentrations wasprepared by diluting a stock solution of bovine serum albumin (BSA). Thestandard solution, blank, and unknown samples (CW, CW_(P), CW_(IO4),PPM, Man, Glu, Glu-B, and Glu-C) were added in 50 μl volumes to thewells of a microtiter plate. The bicinchoninic acid protein assayreagent, BCA (Pierce, USA) was added to each well (200 μl). Thesolutions in the wells were allowed to mix for 30 sec. The plate wasincubated at 37° C. for 45 min. The absorbance was read at 560 nm. Thestandard curve was plotted and the protein concentrations determined foreach sample.

Hexose was determined by the colorimetric method of Dubios et al. withmannose and glucose as standards [Dubios M, Gilles K. A, Hamilton J. K,Robers P. A, Smith S. Colorimetric method for determination of sugarsand related substances. Anal. Chem., 1956. 28: p. 350-356].

The phosphate was determined by the method described by Ames et al. withsome modifications [Ames B. N,. Dubin D. T. The role of polyamines inneutralization of bacteriophage deoxyribonucleic acid. J. Bacteriol.Chem., 1960. 235(3): p. 769-775].

Antibodies to C. albicans

Rabbit antiserum. Antiserum to C albicans was prepared by immunizing tenNew Zealand white rabbits (2-3 kg) with i.v. injections of 200 μl offormalin-killed C. albicans (1 mg/ml, two rabbits) or C albicans CW (1mg/ml) dissolved in PBS. The animals were immunized twice a week foreight weeks. The rabbits were bled once a week prior to the injection ofC. albicans. One week after the last immunization the rabbits wereexsanguinated by heart puncture. Serum was stored frozen. A pool of allten antisera was prepared.

Sera from patients with candidemia. Sera from patients with candidemiawere analyzed for antibody activity against CW, PPM, and Glu by ELISA.Healthy blood donors constituted the control group. The mean age of thepatient and control group was 65±14 and 59.5±4, respectively. The serawere collected from the patients approximately one week after the bloodculturing. All patients were on flucytosine treatment at the time ofblood sampling.

Enzyme-Linked Immunosorbent Assay, ELISA

Microplates (Nunc immunoplate, Denmark) were coated with 100 μl of asuspension of CW, PPM, Man or Glu, diluted in 50 mM Na₂CO₃ buffer, pH9.3. The CW antigen was sonicated prior to coating in order todisaggregate the cell wall fragments which made the solution clearer.Optimal coating concentrations of CW, PPM, Man and Glu were determinedby using the pool of rabbit antiserum diluted to 1/1000. Optimalconcentrations were reached at 50, 5, 5 and 20 μg/ml of CW, PPM, Man andGlu, respectively. The plates were incubated at room temperature for 2h, and thereafter kept at 4° C. overnight. After rinsing the plates oncewith PBS, 100 μl of blocking buffer (1% w/v BSA, 0.05% Tween 20 in PBS)were added to each well and incubated for 1 h at room temperature. Theplates were rinsed once with 0.05% Tween 20 in PBS (PBS-T). Rabbit orhuman serum diluted in tenfold serial steps (1/100-1/10000) in PBS-T,was added to the wells (100 μl) in duplicates and incubated for 2 h atroom temperature. After rinsing the plates three times with PBS-T, 100μl of alkaline phosphatase-conjugated goat anti-rabbit or goatanti-human IgM or IgG were added to the wells. The conjugatedanti-rabbit antibodies (Southern Biotechnology Associates, USA) wereused at dilutions of 1/1000. The conjugated anti-human IgG and IgM(Jackson ImmunoResearch Laboratories, USA) were used in concentrationsof 0.6 μg/ml. After incubation at room temperature for 2 h followed byrinsing, 100 μl of paranitrophenylphosphate (1 mg/ml, Sigma) diluted indiethanolamine buffer (pH 9.8) were added to each well. The absorbancewas read at 405 nm after a suitable color intensity had developed.

Inhibition Assay

Increasing amounts of CW, CW_(IO4), CW_(p), PPM, Man, or the variousglucans (0.1-1000 μg/ml) were added to series of tubes containing aconstant amount of rabbit immune serum. The solutions were incubated at37° C. for 30 min and thereafter at 4° C. overnight. The solutions werecentrifuged to remove any precipitation, and the supernatants wereanalyzed for the remaining antibody activity using CW, Glu, and PPM ascoating antigens. The pool of rabbit immune serum diluted 1/500 (Glu) or1/2000 was used for the inhibition assay. The inhibition capacity of anantigen was defined as the concentration needed for inhibiting theantibody activity to 50%, i.e. reducing the absorbance value to 50% ofthat of the unabsorbed serum dilution (EI₅₀) [Mattsby-Baltzer I,Mielniczuk Z, Larsson L, Lindgren K, Goodwin S. Lipid A in Helicobacterpylori. Infect Immun, 1992. 60(10): p. 4383-7].

Results

Characterization of Antigens

The hexose, protein and phosphate contents of CW, CW_(IO4), CW_(p), PPM,Man, and the glucans are shown in table 1. CW_(IO4) contained the lowestand highest amount of carbohydrate and protein, respecively. This wasexpected since periodate treatment oxidises aldehyde groups ofcarbohydrates [Kanbe T, Morishita M, Ito K, Tomita K, Utsunomiya K,Ishiguro A. Evidence for the presence of immunoglobulin E antibodiesspecific to the cell wall phosphomannoproteins of Candida albicans inpatients with allergies. Clin Diagn Lab Immunol, 1996. 3(6): p. 645-50].The proteinase treatment reduced the protein content to approximately50% of that in CW. PPM contained 10% protein and 0.83% phosphate. Thephosphate content of PPM was approximately 3 and 500 times higher thanin CW and Man respectively. PPM contained 4 times more protein than Man.

SDS-PAGE of PPM revealed a major part of PAS positive material above amolecular weight of 200 kD. Two bands (approximately 48 and 36 kD) wereobserved by protein staining (not shown).

Specificity of Rabbit Antibodies to C albicans

The IgG and IgM antibody responses to CW were analyzed in all immunizedrabbits. The antibody response pattern was almost the same for allrabbits (only five rabbits shown in FIG. 2). The rabbits immunized withwhole yeast cells showed an earlier response. The preimmune sera of therabbits showed no or low antibody levels.

The specificity of the antibodies was further investigated by using CW,CW_(p), CW_(IO4), PPM, Man and Glu as antigens (FIG. 3). The highest IgGantibody levels were observed with PPM. The anti-PPM antibodiescorrelated significantly only with the anti-CW antibodies (P=0.023). TheIgG antibody activity was higher against Man and CW than against themodified CW antigens or Glu. The anti-CW antibodies correlatedsignificantly with the antibodies against proteinase or periodatetreated CW (P=0.010, and 0.028 respectively). The anti-mannan antibodiesdid not correlate with any other antibody. The lowest antibody activitywas found against Glu and CW_(IO4). These antibodies Were stronglycorrelated (P<0.0001). No IgG antibody activity was observed in thepreimmune-sera against the various antigens except for Glu. All rabbitsexcept one showed low anti-Glu antibody activity before the immunization(not shown).

Differences in antibody activity against the varying antigens couldpartly have originated from variations in the binding of the antigens tothe microplate, while still expressing similar or identical antigenepitopes. Therefore cross-reactions between the CW preparations, PPM,Man, and Glu were studied by ELISA inhibition. The pool of rabbit immuneserum was absorbed by CW, CW_(IO4), and CW_(p) and the remainingantibody activity was analyzed by ELISA using CW as antigen (FIG. 4).EI₅₀ for CW_(IO4) was 500 (794 μg/ml) and 50 times (251 μg/ml) higherfor IgM and IgG respectively, compared with the homologous CW antigen(1.6 and 5.4 μg/ml for IgM and IgG, respectively). Thus periodatetreatment of CW reduced the number of antigenic epitopes. The EI₅₀ forCW_(p) was three times higher for IgM and approximately the same for IgG(4 and 5 μg/ml, respectively) in comparison with the homologous antigen,indicating minor influence on antigen epitopes (FIG. 4).

The proportion of anti-CW antibodies directed against PPM, Man, and Gluwas analyzed by absorbing the pooled rabbit immune serum with thecarbohydrate antigens (table 2). EI₅₀ for Glu was approximately 1400-and 16 000 times higher for IgG and IgM compared with PPM. Thecorresponding figures for Man were 5000 and 4000 times. These resultsshowed that the anti-CW antibodies mainly consisted of antibodies to PPMand Man and to a much lesser extent to Glu. Glu-C inhibited the anti-CWIgG antibody activity at 4.5 times higher concentration than needed forGlu.

The IgG antibody activity to Glu after absorption with the homologousantigen, CW, PPM, Man, Glu-B, and Glu-C was also analyzed (Table 3).EI₅₀ was 22 and 28 higher for PPM and CW, respectively, compared withGlu. No inhibition was obtained with Man, Glu-B or Glu-C.

Regarding anti-PPM antibodies, Man and CW inhibited the IgG antibodyactivity to 50% by a concentration 2 and 35 times of that of PPMrespectively, while Glu could not inhibit the antibodies (Table 4).

The changes in antigenic activity of PPM treated by acid or alkali, werecompared with the untreated PPM. The EI₅₀ values for PPM_(HCl) andPPM_(NaOH) were five- and twofold increased compared with untreated PPM.

Antibodies to CW, PPM and Glu in Sera from Patients with Candidemia

Sera from patients with candidemia were compared with the sera fromhealthy blood donors regarding IgG and IgM antibodies to CW, CW_(IO4),PPM and Glu (FIG. 5). The patients showed significantly increased levelsof IgG antibodies to all antigens compared with the control group. TheIgG anti-PPM antibody levels showed the greatest difference between thetwo groups with all patient sera showing higher antibody levels than allthe reference sera. The IgG antibody activity against CW_(IO4) and Gluwere low in both patients and controls. Highly significant correlationswere established between CW and the other antigens (Pearson correlation,P<0.0001). In contrast to the rabbit anti-CW IgG antibodies the humananti-CW IgG antibodies Were highly correlated to the anti-Gluantibodies. Also in humans the anti-CW_(IO4) IgG antibodies correlatedstrongly with anti-Glu antibodies p<0.0001). No significant differenceswere established between the two groups for IgM antibodies.

Discussion

Human serum IgG antibodies to solubilized PPM or as a native cell wallconstituent showed the strongest difference regarding patients withcandidemia and healthy controls. Also IgG antibodies to CW_(IO4) and Gluwere discriminatory. No significantly increased IgM antibody levels wereobserved for any of the antigens suggesting that the C albicansinfection was not a first time challenge. Most probably the patients hadbeen exposed to Candida earlier in life. As a consequence antibody testsusing agglutination would tend to be undiscriminatory, since IgM hasmuch stronger agglutinating capacity than IgG.

PPM, constituted the predominating antigen in the C. albicans cell wallaccording to the antibody analysis of both rabbit immune sera and humansera. The inhibition ELISA revealed no other antigenic cell wallcomponents of significance in the native cell wall except for Man, whichcould be regarded as a chemically stripped form of PPM. Thus, thelocation and abundance of mannoprotein in the outermost layer of thecell wall, is concurrent with high immunogenicity [Nelson R. D, ShibataN, Podzorski R. P, Herron M. J. Candida mannan: chemistry, suppressionof cell-mediated immunity, and possible mechanisms of action. ClinMicrobiol Rev, 1991. 4(1): p. 1-19; Fradin C, Poulain D, Jouault T.beta-1,2-linked oligomannosides from Candida albicans bind to a32-kilodalton macrophage membrane protein homologous to the mammalianlectin galectin-3. Infect Immun, 2000. 68(8): p. 4391-8; Gemmill T. R,Trimble R. B. Overview of N- and O-linked oligosaccharide structuresfound in various yeast species. Biochim Biophys Acta, 1999. 1426(2): p.227-37].

The reported temperature-dependent disappearance of surface antigenicfactors 4, 5, and 6 in C albicans NIH A-207 was not observed with Calbicans strain used by the inventors grown at 37° C. [Okawa Y, TakahataT, Kawamata M, Miyauchi M, Shibata N, Suzuki A, Kobayashi H, Suzuki S.Temperature-dependent change of serological specificity of Candidaalbicans NIH A-207 cells cultured in yeast extract-added Sabouraudliquid medium: disappearance of surface antigenic factors 4, 5, and 6 athigh temperature. FEBS Lett, 1994. 345(2-3): p. 167-71]. Possibleexplanations could be that different strains behave somewhat differentlyor that the growth media are not identical. Whether such surface antigenchanges take place in vivo is not known.

Most immunodiagnostic methods available for routine purposes are basedon the mannan component of the Candida cell wall. Different methods havebeen used for preparation of large amounts of mannan includingextraction with alkali, citrate buffer or water at temperatures of 100to 140° C. [Kocourek J, Ballou C. E. Method for fingerprinting yeastcell wall mannans. J Bacteriol, 1969. 100(3): p. 1175-81, Funayama M,Nishikawa A, Shinoda T, Suzuki M, Fukazawa Y. Antigenic relationshipbetween Candida parapsilosis and Candida albicans serotype B. MicrobiolImmunol, 1984. 28(12): p. 1359-71; Peat S,. Whelan W. J, Edwards. T. E.Polysaccharide of the baker's yeast. Part IV. Mannan. J. Chem. Soc.(London), 1961: p. 29-34; Shibata N, Ichikawa T, Tojo M, Takahashi M,Ito N, Okubo Y, Suzuki S. Immunochemical study on the mannans of Candidaalbicans NIH A-207, NIH B-792, and J-1012 strains prepared by fractionalprecipitation with cetyltrimethylammonium bromide. Arch Biochem Biophys,1985. 243(2): p. 338-48]. Extracted mannan has been separated from othercomponents by precipitation. The solution of Fehling as originallydescribed by Peat et. al. has been used for this purpose in mostserological investigations, although this method has disadvantages[Sendid, B, Tabouret M., Poirot J. L, Mathieu D, Fruit J, Poulain D. Newenzyme immunoassays for sensitive detection of circulating Candidaalbicans mannan and antimannan antibodies: useful combined test fordiagnosis of systemic candidiasis. J Clin Microbiol, 1999. 37(5): p.1510-7; Meckstroth K. L, Reiss E, Keller J. W, Kaufman L. Detection ofantibodies and antigenemia in leukemic patients with candidiasis byenzyme-linked immunosorbent assay. J Infect Dis, 1981. 144(1): p. 24-32;Lehmann P. F, Reiss E. Comparison by ELISA of serum anti-Candidaalbicans mannan IgG levels of a normal population and in diseasedpatients. Mycopathologia, 1980. 70(2): p. 89-93; Au-Young J. K, Troy F.A, Goldstein E. Serologic analysis of antigen-specific reactivity inpatients with systemic candidiasis. Diagn Microbiol Infect Dis, 1985.3(5): p. 419-32]. The alkaline pH of Fehling's solution modifies thestructure of the mannan by cleaving the mannose-serine andmannose-threonine linkages and some peptide bonds. That alkaline pHmodifies the antigen was supported by the increased EI₅₀ value ofPPM_(NaOH) with respect to untreated PPM (table 4). Another disadvantageis that a considerable amount of copper remains bound to the mannan,even after washing the product several times [Nelson R. D, Shibata N,Podzorski R. P, Herron M. J. Candida mannan: chemistry, suppression ofcell-mediated immunity, and possible mechanisms of action. ClinMicrobiol Rev, 1991. 4(1): p. 1-19].

In the study leading to the present invention, PPM was extracted by avery mild method using phosphate buffer (pH 7.0) and a temperature notexceeding 100° C., followed by Cetavlon precipitation. By usingCetavlon, linkages sensitive to cleavage at an alkaline pH werepreserved. PPM contained 10% protein and almost 1% phosphate, which was4- and 400-fold higher compared with the respective concentrations inMan. Based on these chemical data, together with a higher anti-Calbicans antibody activity against PPM than against Man in ELISA, adifferent pattern of the inhibitory activity of PPM compared with Man ininhibition-ELISA, and the significant correlation of anti-PPM to anti-CWantibodies, the preparation procedure according to the invention for PPMappeared to better retain antigenic determinants than the extractionprocedure for Man (FIG. 3, Table 1-4). Possibly critical structures suchas phosphodiester linkages and peptide bonds were left more intact inimmunodominant portions of PPM.

The presence of the charged phosphate groups in the outer region of thewall has effects not only on the attraction or attachment of yeast cellsto other surfaces, but probably also on the antigenicity. The anionicsites on developing germ tubes are believed to be formed by thenegatively charged phosphate groups [Reiss E., Hearn V. M, Poulain D,Shepherd M. G. Structure and function of the fungal cell wall J Med VetMycol, 1992. 30(Suppl 1): p. 143-56]. It has been shown that mutantstrains of C albicans, which lacked the mannosyl-α-phosphoryl andmannosyl-α1-6-linkages, escaped agglutination by a rabbit polyclonalantiserum, raised against C albicans blastoconidia [Whelan W. L, DelgaJ. M, Wadsworth E, Walsh T. J, Kwon-Chung K. J, Calderone R, Lipke P. N.Isolation and characterization of cell surface mutants of Candidaalbicans. Infect Immun, 1990. 58(6): p. 1552-7]. The results suggestthat the phosphate groups and the ester-bound side chains are importantfor antibody recognition. Thus, mannan purification methods that breakup such linkages could be detrimental to the antigen determinants.

Reduction of the protein content in CW, approximately to 50% (Table 1),did not significantly change the antigenicity of CW (FIG. 4). Themodification probably reduced the absorption of the antigen to themicrotiter plate (FIG. 3), since lower antibody activity was observedagainst it than against untreated CW, whereas it retained almostidentical capacity as CW to absorb anti-CW antibodies. Periodateoxidation of CW diminished the antibody activity significantly, probablyas a consequence of reduction in carbohydrate content and furthermodifications of antigenic epitopes. The strong correlation of bothhuman and rabbit IgG anti-CW_(IO4) with anti-glucan antibodies indicatedthat common epitopes occur in both types of preparations, most probablyβ(1-3)(1-6) glucan.

Anti-mannan antibodies are widely and normally found in human sera[Jones J. M. Quantitation of antibody against cell wall mannan and amajor cytoplasmic antigen of Candida in rabbits, mice, and humans.Infect Immun, 1980. 30(1): p. 78-89]. Faux et. al. reported that 76% ofan adult blood donor population had specific antibodies to mannan, andthat the most frequent anti-mannan antibody was of the IgG classdetermined by ELISA [Faux J. A, Agbarakwe A. E, Misbah S. A, Chapel H.M. A comparison of specific IgG antibody levels to the cell wall mannanof Candida albicans in normal individuals and in patients with primaryantibody deficiency. J Immunol Methods, 1992. 153(1-2): p. 167-72]. Themethod for extracting the mannan antigen was, however, not described. Inour study all control sera showed IgG antibody activity to PPM and thePPM-containing native CW. However, the levels of antibody to PPM weresignificantly increased in the patient group. These results are inagreement with the study of Greenfield et. al. who reported that IgGantibodies to mannan (extracted according to Peat et. al.) in ELISA wereincreased during episodes of invasive candidosis in immunosuppressedpatients with a sensitivity of 65% [Greenfield R. A., Bussey, M. J,Stephens J. L, Jones J. M. Serial enzyme-linked immunosorbent assays forantibody to Candida antigens during induction chemotherapy for acuteleukemia. J Infect Dis, 1983. 148(2): p. 275-83]. In a more recent studyin immunocompromised patients anti-mannan antibodies increased withintwo weeks after the probable onset of invasive candidosis in contrast topatients that were only colonized with C albicans [van Deventer A. J,Goessens W. H, van Zeijl J. H, Mouton J. W, Michel M. F, Verbrugh H. A.Kinetics of anti-mannan antibodies useful in confirming invasivecandidiasis in immunocompromised patients. Microbiol Immunol, 1996.40(2): p. 125-31]. A sensitivity of 64% was established in this studyusing a hemagglutination assay, which shows the highest efficiency withIgM antibodies. Since our results show no discriminatory effect of IgMantibodies, improved discrimination would be expected by using IgGantibody specific methods. Au-Young et al. showed that patients withsystemic candidosis had elevated anti-mannan antibodies as tested byELISA compared with patients with bacterial or other fungal infections[Au-Young J. K, Troy F. A, Goldstein E. Serologic analysis ofantigen-specific reactivity in patients with systemic candidiasis. DiagnMicrobiol Infect Dis, 1985. 3(5): p. 419-32]. The interpretation of theresults was difficult, however, since two out of three patients withcandiduria and one out of five controls had anti-mannan antibody levelscomparable to those of the patients with systemic candidosis. In thatstudy a slightly modified version of that of Peat et al. was used forthe preparation of mannan and the antibodies were not analyzed withrespect to their isotypes. Meckstroth et al. concluded that the ELISAtiter of IgG antibody to mannan (prepared according to Peat et. al.)varied widely in leukemic patients irrespective of state ofimmunosuppression or infection and was thus of no diagnostic value[Meckstroth K. L, Reiss E, Keller J. W, Kaufman L. Detection ofantibodies and antigenemia in leukemic patients with candidiasis byenzyme-linked immunosorbent assay. J Infect Dis, 1981. 144(1): p.24-32]. In view of our results and the reports using the mannan forantibody IgG analyses by ELISA, the specificity seems to improve withless modified mannan antigens such as our PPM which might be furtherenhanced with subclass IgG antibody analysis.

In contrast to PPM and Man, glucan with β(1-3)(1-6)D linkages was lesseffective in inhibiting the anti-CW antibody, suggesting thatanti-glucan antibodies were not a major component in the rabbit immuneserum. In addition, the anti-Glu antibodies were highly specific forβ(1-3)(1-6) linkages, since no inhibitory activity was observed byβ(1-4)(1-6) or β(1-3) glucan. The anti-Glu levels in humans withcandidemia were as low as for CW_(IO4). Since β(1-3)(1-6) glucan aloneinduces high antibody levels in immunized rabbits (authors unpublishedresults) our results are in agreement with the suggestion that glucan islocated deeper in the cell wall of C. albicans and thereby lessimmunogenic [Sanjuan R, Zueco J, Zueco J, Perez J, Penarroja C,Sentandreu R. A comparative study of the incorporation of a1,6-beta-glucan and an O-glycosylated protein epitope into the cell wallof Candida albicans. Microbiology, 1996. 142(Pt 8): p. 2255-62]. Astronger immunogenicity would probably require breakdown of the cellwall fragments and presentation by macrophages.

In summary, IgG antibodies to PPM and the native CW were found to bepotential markers for detection of invasive Candida infection.

EXPERIMENTAL—PART II Material and Methods

Study Group

Candidiasis. Serum samples were collected on two to three occasionsafter documentation of systemic candidiasis by culturing from 14patients (mean age 62±12 years) (Table 2). The following criteria wereapplied when laboratory and clinical files were examined: (i) positiveculture of specimens from normally sterile sites (blood, bile, drain,pericardial fluid); (ii) the presence of risk factors (cancer andchemotherapy, abdominal surgery or use of broad-spectrum antibiotics);(iii) the presence of infectious syndrome (fever) that did not respondto antibacterial therapy.

Controls. Two control groups were included in this study. One group(group I) comprised nine lactating mothers with superficial C. albicansinfection of the nipples (mean age 31±5) and ten healthy blood donors (8men and 2 women, mean age 53±7.5) (group II).

IgG Subclass Antibody Analysis.

Microplate wells (Nunc immunoplate, Denmark) were coated with 100 μl of50 μg/ml of CW or CW_(IO4). For glucan and PPM 20 and 5 μg/mlrespectively were used. The antigens were diluted in 50 mM Na₂CO₃buffer, pH 9.3. The plates were incubated at room temperature (r.t.) fortwo hours and thereafter kept at 4° C. overnight. After rinsing theplate once with PBS, 100 μl of blocking buffer (BF) (1% BSA, 0.05% Tween20 in PBS) was added to each well and the plate incubated for 1 h atr.t. The plate was rinsed once with 0.05% Tween-20 in PBS (PBS-T). Humansera diluted in tenfold serial steps (1/100-1/10000) in BF were added toeach well (100 μl) and incubated for two hr at r.t. Hereafter the plateswere rinsed three times with PBS-T between each incubation step. Murinemonoclonal antibodies (Immunotech) to IgG1, (JL512), IgG2 (GOM1), IgG3(ZG4) and IgG4 (RJ4) diluted in BF were added to the wells (100 μl). Theplate was incubated at r.t. for 2 h, and thereafter 100 μl ofbiotinylated rabbit anti mouse IgG (Dako, Denmark) diluted 1/5000 in BFwere added. After incubation at r.t. overnight, 100 μl of alkalinephosphates conjugated extravidin (Sigma) diluted 1/10000 were added andthe plate was incubated at r.t. for 60 min. Para-nitrophenylphosphate (1mg/ml, Sigma, USA) diluted in diethanolamin buffer (pH 9.8) was added toeach well and the absorbance was read at 405 nm when a suitable colorhad developed.

The antibody titer of a serum was defined as the log of dilution thatgave an absorbance value of 0.15 above the background value. Two serumsamples were included in each assay as standards, one with a highantibody titer (pooled sera from patients) and another one with a lowantibody titer (pooled sera from healthy individuals). If thesestandards deviated more than 10% from their values, the results wereadjusted accordingly.

Glucan determination. The β(1-3)-glucan concentration in serum wasdetermined by a commercial kit, Gluspecy (Seikagaku, Japan). Allglasswares were heated to 180° C. for 4 h in order to remove anycontaminants. Serum samples were diluted 1/10 in pyrogen free water andheat inactivated at 75° C. for 10 min in order to inactivate inhibitoryfactors possibly present in the serum (ADD REF). Serum and standardsamples were added to the microplate wells (50 μl) before pippeting 50μl of lysate reagent into the wells. The plate was incubated at 37° C.for 30 min, then 50 μl of each of the azo-coupling reagents were addedto the wells. The azo-coupling increased the sensitivity of the assay.The absorbance was read at 560 nm. The standard curve was plotted andβ(1-3)-glucan concentration determined for each sample. The detectionlimit of the assay was 1 μg/ml and accordingly the detection limit was10 μg/ml of the serum samples.

Mannan determination. For determination of mannan in serum acommercially available latex agglutination test Candida-Pastorex (SanofiDiagnostics Pasteur) was used. The assay was run according to themanufacture's instruction. To 300 μl of patient serum were added 100 μlof EDTA treatment solution and the mixture boiled for 3 min andcentrifuged at 10 000×g for 10 min. Ten μl of Candida latex solution wasadded to 40 μl of supernatant. The solution was placed on a mixer for 10min and thereafter examined for agglutination.

Statistical analysis. Data were analyzed by the method of Kruskal-Wallisto avoid random significance when comparing several groups. Correlationswere analyzed by spearman's rank correlation test.

Results

Circulating Antigens

Analysis of patients with candidiasis showed that 37 out of all 38samples were positive (>20 pg/ml) for β(1→3)-glucan (FIG. 6). Theβ(1-3)-glucan negative sample was however collected from the patient(patients 12) at least three weeks after the first cultivation of C.tropicalis from the patients (FIG. 6). Only one (patient 7) out of fivepatients (7, 8, 9, 10, 11) who succumbed to the Candida infection showedglucan concentrations below 100 pg/ml in all samples. The other fourpatients had levels of at least 120 pg/ml (FIG. 6). The other patientswho survived had glucan levels lower than 100 pg/ml in their last serumsample except four of them (patient 1, 4, 5, 14). However, for one ofthese patients the glucan levels dropped in from over 300 to 110 pg/ml,probably indicating a succesful recovery. The patient with the lowestglucan levels (no, 12, Table 1) were infected with C. tropicalis.

The concentrations of β(1→3)-glucan in serum samples from patients withsuperficial fungal infection and from the healthy blood donors were allbelow 20 pg/ml (FIG. 6). Thus the concentration of β(1→3)-glucan inserum samples from patients with invasive candidiasis was significantlyelevated compared to those in controls.

Patient and controls were also analysed for mannan by Pastorex Candidaassay. All of the patients and controls were negative.

IgG Subclass Antibodies Against C albicans Cell Wall Antigens.

CW. The IgG subclass antibody analysis using C albicans CW as antigenshowed that IgG1, IgG2, and IgG3 antibodies were significantly elevatedin patients compared to the control groups (FIGS. 7 a, b, and c). Allsamples from two out of four patients (no. 12 and 14, Table 2) infectedwith C. tropicalis and C. glabrata were negative for IgG1 antibody to Calbicans CW (FIG. 8 a). The highest antibody titers were found withinthe first week (<7) of culture-proven candidiasis. The titer of C.albicans infected patients were significantly higher than those ofnon-albicans infected ones for IgG1 antibodies to CW first after 14 daysor more (p=0.049, not shown). Only one of the superficially Candidainfected women in group I was positive for IgG1 antibodies to CW (FIG. 7a).

Three patients were negative for IgG2 antibodies to CW (FIG. 8 b). Twoout of these patients were infected with C. glabrata (FIG. 8 b, Table1). Two out of nine in control group I were shown positive values closeto the border line for this antibody (FIG. 7 b).

Regarding IgG3 antibodies to CW, one of the two patients who werenegative was infected with C. glabrata (FIG. 8 c). The highest IgG3antibody titers were found between 7-13 days after positive bloodcultures (FIG. 7 c). All the healthy blood donors were negative for IgG3antibodies, while two women with superficial Candida infection in groupI were positive. No differences (P>0.05) were found between the patientsand the controls regarding IgG4 and IgM antibodies to CW. The ELISAtiter were usually very similar in patients as well as controls (datanot shown).

CW_(IO4), glucan, and PPM. Concerning IgG1, IgG3 and IgG4 antibodies toCW_(IO4), glucan, and PPM no discriminatory differences were foundbetween the patients and the controls (data not shown). Differencesbetween patients and controls were found, however for IgG2 antibodies tothese antigens (FIG. 7 d, e, f and Table 3).

For the two last sampling points in patients (≧7-13, and ≧14) the levelsof IgG2 antibodies to CW_(IO4) were significantly higher when comparedwith control group I (FIG. 7 e). Four patients were negative, and one ofthese did not survive (FIG. 8 e). In addition, all four presentedincreased glucan values with time (P=0.0037, Fischer's exact test).

IgG2 antibodies to PPM were significantly increased in patients comparedto the controls (FIG. 7 d, table 3). All of the patients were positivefor IgG2 antibody to PPM (FIG. 8 d). Only one serum sample was negativewithin the first week. This very early serum sample was negative inother antibody tests. However, also three controls in group I werepositive for IgG2 antibodies to PPM, while all of the healthy blooddonors were negative (FIG. 7 d). Looking at the ratios of IgG1anti-CW/IgG2 anti-PPM they were significantly lower in non-survivingpatients than in the other patients, especially within the first week(<7) after the cultivation of Candida from blood (P=0.019, not shown).

IgG2 antibodies to glucan were also significantly higher compared to thecontrols except when comparing the serum samples of <7 days (FIG. 7 f).Three patients, all infected with C albicans, were negative at all timepoints and were the same as found negative for IgG anti-CW_(IO4)antibodies (FIG. 8 f). One of the controls in group I and another one ingroup II were positive (FIG. 7 f).

A strong correlation was found between IgG2 antibodies to CW and PPM(Table 3), and between IgG2 antibodies to CW_(IO4) and glucan (P<0.0001,not shown) reflecting the predominating PPM antigen in CW and the glucancontent of CW_(IO4), respectively. No correlation was observed betweenIgG1 anti-CW and the IgG2 anti-CW_(IO4), -PPM, and -glucan antibodiesindicating that the IgG1 antibodies are directed against other antigenicepitopes in the CW (Table 3).

No significant correlation was found between glucan concentration inserum and any IgG subclass antibody. However glucan correlated with IgMantibodies to CW (P<0.0001).

The sensitivity, specificity, positive and negative predictive values ofthe antibody detection tests were calculated (Table 5). The sensitivityof IgG2 antibody detection test increased from 85% for CW to 98% whenPPM was used. The sensitivity of IgG1 and IgG3 antibodies to CW werealmost the same (80% and 85% respectively). The lowest specificity wasfound for IgM antibodies to CW, which makes these antibodies of nodiagnostic value.

It is important to be able to distinguish patients with systemiccandidiasis from other infections as early as possible after the onsetof clinical symptoms. For this reason the discriminatory power of thecombination of the most sensitive method (IgG2 anti-PPM) with the mostspecific one (IgG1 anti-CW antibody) was analyzed (Table 4). A positiveserum sample was defined as either a titer of log 3 for any of the twoantibodies or a log sum of at least 5. Although the sensitivity was 70%within the first week (<7) of culture-proven candidiasis, thecombination of these antibodies increased the specificity and positivepredictive value to 100%. Although the glucan concentration on its ownidentified all candidiasis cases, this parameter together with thecombined IgG2 anti-PPM and IgG1 anti-CW antibodies strongly support adeep candida infection. TABLE 1 Chemical analysis of C. albicans cellwall preparations, PPM, Man, and glucans. Amount, % of dry weightAntigens Hexose Protein Phosphate CW 93 16 0.27 CW_(IO4) 83 (60=) 0.84CW_(p) 90 8 0.11 PPM 95 10 0.83 Man 99 2.4 0.002 Glu 100 6 0.03 Glu-B100 0 0.10 Glu-C 100 0 ND**Not determined.=The CW_(IO4) was not soluble in the protein assay and thus theabsorbance showed too high value due to turbidity.

TABLE 2 Underlying diseases and culture data of patients indicated inthe systemic candidiasis. No of Candida Patient Age Hospital Underlyingserum Cultured Candida no Sex* (yr) ward Condition specimens fromSpecies Outcome 1 F 71 ICU Rectal neoplasms 3 Blood C. albicans survived2 F 67 Surgery Pancreaticoduodenectomy 3 Blood C. albicans survived 3 F40 Neurology Aneurysm 3 Blood survived 4 M 74 ICU Aortic aneurysm 3Blood C. albicans survived 5 M 61 ICU Diabetes mellitus 3 Blood C.albicans survived hemorrhagic pancreatitis 6 F 56 E*** E 3 Blood C.albicans survived 7 F 74 ICU Diabetes mellitus 3 Blood C. albicans >** 8F 50 Surgery Non-Hodgkin's lymphoma 3 Blood C. albicans > 9 F 47 ICUNeoplasm, Intestinal 3 Blood C. albicans > obstruction 10 M 66Transplantation Diabetic angiopathies and 3 Abdominal C. albicans > Unitnephropathies 11 M 66 Thorax Atrial fibrillation 3 Blood C. albicans >12 F 82 Transplantation Gallbladder neoplasms 3 Blood C. tropicalissurvived Unit 13 M 64 Surgery Diabetic coma 2 Blood/Drain C. glabratasurvived 14 F 46 Transplantation Hepatitis B and 3 Bile C. glabratasurvived Unit hepatitis C*M, male; F, female.**>Dead***Epatient was described with Candida septicaemia. (sepsis)

TABLE 3 The correlation between IgG2 antibodies in serum sample frompatients with systemic candidiasis. The correlation was calculated bySpearman's rank correlation test. IgG2 CW CW_(IO4) PPM Glucan CW-IgG1 nsns ns ns CW-IgG2 — 0.0010 <0.0001 ns CW-IgG3 0.0372 0.0191 0.0484 0.0077CW-IgM 0.0069 ns 0.0317 ns

TABLE 4 Sensitivity, specificity and predictive values for the detectionof subclass antibodies in patients with systemic candidiasis. Parameters^(a) CW CW CW CW PPM CW_(IO4) Glucan % IgG1 IgG2 IgG3 IgM IgG2 IgG2 IgG2Sensitivity 80 85 85 90 98 66 61 Specificity 95 89 89 26 95 74 89Positive 97 94 95 74 93 85 93 Predictive Value Negative 69 74 74 56 9550 52 Predictive Value^(a) Results are calculated per serum according to the analysis of 41serum samples from 14 patients with systemic candidiasis, and 19 serumsamples from controls.

TABLE 5 Sensitivity, specificity, and predictive values for combineddetection of IgG1 anti-CW and IgG2 anti-PPM antibodies at different timepoints after culture-proven Candidiasis. Combination of IgG1 anti-CW andIgG2 anti-PPM* Parameters ^(a) <7 days 7-13 days >14 days Sensitivity 7092 85 Specificity 100 100 100 Positive predictive value 100 100 100Negative predictive value 86 95 90^(a) Results are calculated per serum according to the analysis of serumsamples from patients with systemic candidiasis, and 19 serum samplesfrom controls.*In order to be positive the titer of any of the two antibodies had tobe either equal to or exceed log 3, or the combined values equal to ormore than log 5.Discussion

The diagnosis of invasive candidiasis is extremely difficult, bothclinically and microbiologically (Jones, J. M. 1990. Laboratorydiagnosis of invasive candidiasis. Clin Microbiol Rev 3:32-45). To solvethis problem, serological diagnostic methods based on detection ofmarker substances as well as antibodies to Candida have been used (deRepentigny, L., R. J. Kuykendall, F. W. Chandler, J. R. Broderson, andE. Reiss. 1984. Comparison of serum mannan, arabinitol, and mannose inexperimental disseminated candidiasis. J Clin Microbiol 19:804-12;Gutierrez, J., C. Maroto, G. Piedrola, E. Martin, and J. A. Perez. 1993.Circulating Candida antigens and antibodies: useful markers ofcandidemia. J Clin Microbiol 31:2550-2; Obayashi, T., M. Yoshida, T.Mori, H. Goto, A. Yasuoka, H. Iwasaki, H. Teshima, S. Kohno, A.Horiuchi, A. Ito, and et al. 1995. Plasma (1-->3)-beta-D-glucanmeasurement in diagnosis of invasive deep mycosis and fungal febrileepisodes. Lancet 345:17-20; Reiss, E., T. Obayashi, K. Orle, M. Yoshida,and R. M. Zancope-Oliveira. 2000. Non-culture based diagnostic tests formycotic infections. Med Mycol 38:147-59). Some of these techniques arecommercially available, but their clinical reliability of these productsis still controversial. In the present study glucan determination andanalysis of IgG1 anti-CW and IgG2 anti-PPM antibodies were found to bereliable and early markers of systemic candidiasis. All patients withinthe first two weeks of systemic candidiasis were positive for glucan.The glucan concentrations in all controls were below the cut-off value(<20 pg/ml). Likewise, patients with IgG1 anti-CW or IgG2 anti-PPM witha high titer (≧log 3), or the combined titer of the antibodies(titer≧log 5) showed 92% sensitivity and 100% specificity.

These results show the potential value of antibodies in the laboratoryassessment of patients with systemic candidiasis, which is in agreementwith others (Sendid, B., M. Tabouret, J. L. Poirot, D. Mathieu, J.Fruit, and D. Poulain. 1999. New enzyme immunoassays for sensitivedetection of circulating Candida albicans mannan and antimannanantibodies: useful combined test for diagnosis of systemic candidiasis.J Clin Microbiol 37:1510-7; van Deventer, A. J., W. H. Goessens, J. H.van Zeijl, J. W. Mouton, M. F. Michel, and H. A. Verbrugh. 1996.Kinetics of anti-mannan antibodies useful in confirming invasivecandidiasis in immunocompromised patients. Microbiol Immunol 40:125-31).With our CW and PPM antigens all patients with systemic candidiasisexcept one were positive in their antibody levels within the first weekof their infection. The highest IgG2 anti-PPM antibodies were foundwhithin 7-13 days after cultivation of Candida, which is in agreementwith van Deventer et al. who observed that anti-mannan antibody titer inimmnuocompromised patients with invasive candidiasis increased withintwo weeks after the possible onset of invasive candidiasis. (vanDeventer, A. J., W. H. Goessens, J. H. van Zeijl, J. W. Mouton, M. F.Michel, and H. A. Verbrugh. 1996. Kinetics of anti-mannan antibodiesuseful in confirming invasive candidiasis in immunocompromised patients.Microbiol Immunol 40:125-31). Sendid et al. showed that combined testfor detection of circulating mannan and anti-mannan antibodies is usefulfor detection of patients with systemic candidiasis, with thesensitivity and specificity of 80 and 93% which were calculated perpatient, when at least one serum was positive by one of the tests.Sendid et al. demonstrated that seum samples with high concentration ofcirculating mannan had a low level of antimannan antibodies and viceversa (Sendid, B., M. Tabouret, J. L. Poirot, D. Mathieu, J. Fruit, andD. Ponlain. 1999. New enzyme immunoassays for sensitive detection ofcirculating Candida albicans mannan and antimannan antibodies: usefulcombined test for diagnosis of systemic candidiasis. J Clin Microbiol37:1510-7). The inventors did not find any assosiation of circulatingglucan and IgG2 anti-glucan, PPM and CW in serum. However, a verysignificant correlation was found between circulationg glucan and IgManti-CW antibodies.

Patients, who did not survive the fungal infection had significantlylower ratios of IgG1 anti-CW to IgG2 anti-PPM antibodies during thefirst week after isolation of Candida. That high concentration of IgG2antibodies may interfere with binding of IgG1 or IgG3 and therebydiminishing or preventing the bacteria from being properly phagocytosedhas been suggested for patients with high IgG2 antibodies to P.aeruginosa in cystic fibrosis (Pressler, T., B. Mansa, T. Jensen, S. S.Pedersen, N. Hoiby, and C. Koch. 1988. Increased IgG2 and IgG3concentration is associated with advanced Pseudomonas aeruginosainfection and poor pulmonary function in cystic fibrosis. Acta PaediatrScand 77:576-82).

No consistent difference was found in the different antibody responsesto the C. albicans cell wall antigens between patients infected with C.albicans and those infected with other Candida species. This is notsurprising, since mannans of different Candida species have similarstructures and share common epitopes, which cross-react with antibodiesto C. albicans (Suzuki, S. 1997. Immunochemical study on mannans ofgenus Candida. 1. Structural investigation of antigenic factors 1, 4, 5,6, 8, 9, 11, 13, 13b and 34. Curr Top Med Mycol 8:57-70). The inventorshave observed that serum from rabbits immunized with C. albicanscross-reacts with PPM extracted from C. glabrata and C. parapsilosis.However out of the seven altogether negative anti-CW tests encompassingIgG1, IgG2 and IgG3 antibodies five represented all the patientsinfected with non-albicans Candida species. This might be a result ofspecies differences with respect to CW antigens. Furthermore, failure ofanti-CW IgG1, IgG2 or IgG3 antibody response may be an indication to useanti-fungal treatment covering non-albicans species, whereas when thetiters of all these substances are significant anti-albicans treatmentshould suffice.

The time required for diagnosis of systemic candidiasis is an importantfactor in decreasing the mortality among these patients. Thus, a highpositive predictive value of the glucan and antibody analyses at theonset of systemic candidiasis is of help when initiating an earlytreatment by antifungal. The treatment of patients with invasive fungalinfections by antifungal agents, however, is limited and does not differmuch with the fungal species. It may therefore be more practical toanalyse a blood sample for fungal antigens such as β(1→3)-glucan andantibodies first and if these parameters are positive start antifungaltherapy immediately, while searching for fungal species. By thisstrategy the mortality among patients with invasive fungal infectionsmay be decreased.

In conclusion, our results suggest that the combination of IgG2anti-PPM, IgG 1 and IgG3 anti CW constitute a reliable laboratory teststo diagnose patients with systemic candidiasis in an early stage.

1. A method for the diagnosis of candidiasis or invasive candidiasiscomprising assaying with a combination of an IgG2 antibody to aphosphopeptidomannan (PPM) fraction of the cell wall of C albicans, andan IgG1 antibody to a C albicans cell wall antigen, and glucan.
 2. Amethod for the diagnosis of candidiasis or invasive candidiasiscomprising assaying with an antibody to a C albicans cell wall antigenor to a solubilized phosphopeptidomannan (PPM) fraction of the cell wallof C albicans.
 3. A method according to claim 2, wherein said antibodyis an IgG2 antibody.
 4. A method according to claim 2, wherein saidantibody is an IgG1 antibody.
 5. A method according to claim 2, whereinsaid antibody is an IgG3 antibody.
 6. Diagnostic kit for the diagnosisof candidiasis or invasive candidiasis comprising means for drawing asample from a patient; means for an assay for the detection of acombination of an IgG2 antibody to a phosphopeptidomannan (PPM) fractionof the cell wall of C albicans, and an IgG1 antibody to a C albicanscell wall antigen, and glucan, wherein said sample is analyzed for thepresence of the simultaneous presence of an IgG2 antibody to aphosphopeptidomannan (PPM) fraction of the cell wall of C albicans, andan IgG1 antibody to a C albicans cell wall antigen, and glucan.
 7. Thediagnostic kit according to claim 6, wherein said assay is a sandwichELISA assay.
 8. Diagnostic kit for the diagnosis of candidiasis orinvasive candidiasis comprising means for drawing a sample from apatient; means for an assay for the detection of an antibody to a Calbicans cell wall antigen or to a solubilized phosphopeptidomannan(PPM) fraction of the cell wall of C albicans, wherein said sample isanalyzed for the presence of an antibody to a C albicans cell wallantigen or to a solubilized phosphopeptidomannan (PPM) fraction of thecell wall of C albicans.
 9. The diagnostic kit according to claim 8,wherein said antibody is an IgG2 antibody.
 10. The diagnostic kitaccording to claim 8, wherein said antibody is an IgG1 antibody.
 11. Thediagnostic kit according to claim 8, wherein said antibody is an IgG3antibody.
 12. The diagnostic kit according to claim 8, wherein saidassay is a sandwich ELISA assay.
 13. A method for diagnosing candidiasisor invasive candidiasis in a patient comprising drawing a sample fromthe patient, and performing an assay for the detection of an IgG2antibody to a phosphopeptidomannan (PPM) fraction of the cell wall of Calbicans, and an IgG1 antibody to a C albicans cell wall antigen, andglucan, wherein the simultaneous presence of an IgG2 antibody to aphosphopeptidomannan (PPM) fraction of the cell wall of C albicans, andan IgG1 antibody to a C albicans cell wall antigen, and glucan indicatescandidiasis or invasive candidiasis in the patient.
 14. A method fordiagnosing candidiasis or invasive candidiasis in a patient comprisingdrawing a sample from the patient, and performing an assay for thedetection of an antibody to a C albicans cell wall antigen or to asolubilized phosphopeptidomannan (PPM) fraction of the cell wall of Calbicans, wherein the presence of an antibody to a C albicans cell wallantigen or to a solubilized phosphopeptidomannan (PPM) fraction of thecell wall of C albicans indicates candidiasis or invasive candidiasis inthe patient.
 15. A method for the diagnosis of candidemia or invasiveCandida infection comprising assaying with an antibody.
 16. A methodaccording to claim 15, wherein said antibody is an IgG antibody to anative cell wall fragment of C albicans.
 17. A method according to claim15, wherein said antibody is an IgG antibody to a solubilizedphosphopeptidomannan (PPM) fraction of the cell wall of C albicans. 18.A method according to claim 16, wherein said IgG antibody is a humanserum IgG antibody.
 19. Diagnostic kit for the diagnosis of candidemiaor invasive Candida infection comprising means for drawing a sample froma patient; means for an assay for the detection of an IgG antibody to anative cell wall fragment of C albicans or an IgG antibody to asolubilized phosphopeptidomannan (PPM) fraction of the cell wall of Calbicans, wherein said sample is analyzed for the presence of an IgGantibody to a native cell wall fragment of C albicans or an IgG antibodyto a solubilized phosphopeptidomannan (PPM) fraction of the cell wall ofC albicans.
 20. The diagnostic kit according to claim 19, wherein saidassay is a sandwich ELISA assay.
 21. The diagnostic kit according toclaim 19, wherein said antibody is a human serum IgG antibody.
 22. Amethod for diagnosing candidemia or invasive Candida infection in apatient comprising drawing a sample from the patient, and performing anassay for the detection of an IgG antibody to a native cell wallfragment of C albicans or an IgG antibody to a solubilizedphosphopeptidomannan (PPM) fraction of the cell wall of C albicans,wherein the presence of an IgG antibody to a native cell wall fragmentof C albicans or an IgG antibody to a solubilized phosphopeptidomannan(PPM) fraction of the cell wall of C albicans indicates candidemia orinvasive Candida infection in the patient.
 23. The method according toclaim 22, wherein said antibody is a human serum IgG antibody.
 24. Thediagnostic kit according to claim 9, wherein said assay is a sandwichELISA assay.
 25. The diagnostic kit according to claim 10, wherein saidassay is a sandwich ELISA assay.
 26. The diagnostic kit according toclaim 11, wherein said assay is a sandwich ELISA assay.
 27. A methodaccording to claim 17, wherein said IgG antibody is a human serum IgGantibody.
 28. The diagnostic kit according to claim 20, wherein saidantibody is a human serum IgG antibody.